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de Jonge, R., van Esse, H.P., Maruthachalam, K., Bolton, M.D., Santhanam, P., Saber,. 698 ..... Schaible, L., Cannon, O.S., and Waddoups, V. (1951). Inheritance ...
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The plant specific transcription factors CBP60g and SARD1 are targeted by a

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Verticillium secretory protein VdSCP41 to modulate immunity

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Jun Qin1, Kailun Wang1,2, Lifan Sun1, Haiying Xing1, Sheng Wang1,2, Lin Li3, She

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Chen3, Hui-Shan Guo1,2* and Jie Zhang1,4*

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State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese

Academy of Sciences, Beijing, 100101, China. 2

University of Chinese Academy of Sciences, Beijing 100049, China.

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National Institute of Biological Sciences, Beijing, China.

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Lead Contact

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Corresponding author: Jie Zhang and Hui-Shan Guo

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Correspondence should be addressed to: Jie Zhang ([email protected]) or Hui-Shan

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Guo ([email protected])

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Tel: 8610-64806355.

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Key Words: Verticillium, effector, calmodulin-binding protein, plant immunity

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ABSTRACT 1

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The vascular pathogen Verticillium dahliae infects the roots of plants to cause

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Verticillium wilt. The molecular mechanisms underlying V. dahliae virulence and host

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resistance remain elusive. Here, we demonstrate that a secretory protein, VdSCP41,

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functions as an intracellular effector that promotes V. dahliae virulence. The

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Arabidopsis master immune regulators CBP60g and SARD1 and cotton GhCBP60b

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are targeted by VdSCP41. VdSCP41 binds the C-terminal portion of CBP60g to

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inhibit its transcription factor activity. Further analyses reveal a transcription

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activation domain within CBP60g that is required for VdSCP41 targeting. Mutations

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in both CBP60g and SARD1 compromise Arabidopsis resistance against V. dahliae

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and partially impair VdSCP41-mediated virulence. Moreover, Virus-induced silencing

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of GhCBP60b compromises cotton resistance to V. dahliae. This work uncovers a

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virulence strategy in which the V. dahliae secretory protein, VdSCP41, directly targets

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plant transcription factors to inhibit immunity, and reveals CBP60g, SARD1 and

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GhCBP60b as crucial components governing V. dahliae resistance.

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INTRODUCTION The vascular pathogen Verticillium dahliae infects a broad range of plants and 2

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causes devastating diseases. V. dahliae can survive in the form of microsclerotia in

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soil for over ten years (Schnathorst, 1981). During V. dahliae colonization,

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microsclerotia germinate and develop hyphae that adhere tightly to the root surface

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upon the perception of plant roots (Zhao et al., 2014). A few hyphae form hyphopodia

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at the infection site and further differentiate into penetration pegs to penetrate into

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plant cells and colonize the vascular tissue (Fradin and Thomma, 2006; Schnathorst,

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1981; Vallad and Subbarao, 2008; Zhao et al., 2014; Zhao et al., 2016). Although a

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few key steps mediating these infection processes have been elucidated, the molecular

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mechanisms underlying V. dahliae virulence remain largely unknown.

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Progress has been made in isolating virulence factors that are crucial for V.

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dahliae virulence. Several genes that regulate V. dahliae development have been

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characterized as contributing to virulence. VDH1 and VdGARP1, which regulate

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microsclerotial development, are required for V. dahliae virulence in cotton plants

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(Gao et al., 2010; Klimes and Dobinson, 2006). VdRac1 and VdPKAC1 regulate both

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the development and pathogenicity of the fungus in host plants (Tian et al., 2015;

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Tzima et al., 2010; Tzima et al., 2012). VdEG1, VdEG3, VdCYP1, and VdSNF1 also

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function as factors that contribute to V. dahliae virulence on cotton (Gui et al., 2017;

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Tzima et al., 2011; Zhang et al., 2016).

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In addition to development-associated virulence factors, fungal pathogens deliver

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effectors that act as virulence factors, inhibiting host defense to promote pathogenesis,

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which also occurs in bacterial and oomycete pathogens. The race 1 strain-specific

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effector Ave1 contributes to V. dahliae virulence on tomato plants not carrying Ve1 (de 3

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Jonge et al., 2012). Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins

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(NLPs) (NLP1 and NLP2) secreted by V. dahliae strain JR2 are required for its

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pathogenicity in tomato and Arabidopsis plants (Santhanam et al., 2013). VdSge1

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encodes a transcriptional regulator that controls the expression of six putative effector

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genes. Deletion of VdSge1 in V. dahliae results in significantly impaired pathogenicity

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in tomato, suggesting an important role of secreted effectors in suppressing host

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immunity in V. dahliae (Santhanam and Thomma, 2013).

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However, only two secreted effectors of V. dahliae have been indicated to

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function inside the plant cell to modulate host immunity thus far. VdIsc1, a V. dahliae

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effector lacking a known signal peptide, is thought to be delivered into host cells to

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hydrolyze a salicylic acid (SA) precursor and inhibit salicylate metabolism (Liu et al.,

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2014). The small cysteine-containing protein (SCP) VdSCP7 translocates into the

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nucleus of plant cells to either suppress or induce defense in plants through unknown

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mechanisms (Zhang et al., 2017).

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Plants are equipped with immune components to counteract V. dahliae virulence.

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Tomato Ve1 was identified as an effective resistance locus that recognizes Ave1

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secreted by Race 1 strains (Fradin et al., 2009; Schaible et al., 1951). Genetic analyses

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indicated that EDS1, NDR1 and SERK3/BAK1 are required for Ve1-mediated

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resistance in both tomato and Arabidopsis (Fradin et al., 2011; Fradin et al., 2009).

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Studies in cotton also showed that GhBAK1 and GhNDR1 are crucial components

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regulating defense against V. dahliae (Gao et al., 2013b; Gao et al., 2011),

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demonstrating the common requirements of NDR1 and SERK3/BAK1 in a conserved 4

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defense mechanism against V. dahliae in these plants. GbWRKY1, GhSSN, GbERF,

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GhMLP28, GhMKK2 and GbNRX1 have also been shown to be required for cotton

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resistance against V. dahliae (Gao et al., 2011; Li et al., 2014a; Li et al., 2016; Qin et

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al., 2004; Sun et al., 2014; Yang et al., 2015). A comparative proteomic analysis

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indicated the involvement of both brassinosteroids and jasmonic acid signaling

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pathways in the regulation of cotton resistance to V. dahliae (Gao et al., 2013a).

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Although several regulators have been identified, the mechanisms by which

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plants defend against V. dahliae remain obscure, and further investigation is required

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to isolate more host immune components governing V. dahliae resistance. Targeting

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key immune components is a common strategy employed by pathogenic effectors to

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promote pathogenicity (Boller and He, 2009; Cui et al., 2009; Dou and Zhou, 2012);

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thus, screening of host proteins targeted by pathogenic effectors provides an efficient

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way to identify crucial host defense components.

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The calmodulin-binding proteins (CBPs) functions to bind calmodulin to

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transduce calcium signals (Bouche et al. 2005). The plant-specific CALMODULIN

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BINDING PROTEIN 60 (CBP60) protein family contains eight family members

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including CBP60a–g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1

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(SARD1) (Bouche et al. 2005). CBP60g and SARD1 are two most closely related

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members that function partially redundantly in both SA signaling and bacterial

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resistance (Zhang et al. 2010; Wang et al., 2011). CBP60g contains a

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calmodulin-binding domain (CBD) which is essential for its function in defense,

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whereas SARD1 does not bind calmodulin (Wang et al., 2009; Wang et al. 2011; 5

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Zhang et al. 2010). Both CBP60g and SARD1 function as transcription factors that

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directly bind to promoters of genes controlling SA synthesis, such as

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ISOCHORISMATE SYNTHASE 1 (ICS1), ENHANCED DISEASE SUSCEPTIBILITY

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5 (EDS5), NON-EXPRESSOR OF PATHOGENESIS RELATED GENES1 (NPR1)

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(Dong et al., 2004; Nawrath et al., 2002; Sun et al., 2015; Wildermuth et al., 2004).

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Moreover, ChIP-seq analyses have revealed the binding of CBP60g and SARD1 to

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the promoters of a number of genes regulating pathogen-associated molecular patterns

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(PAMPs)-triggered immunity (PTI), effector-triggered immunity (ETI) and systemic

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acquired resistance (SAR) (Sun et al., 2015), indicating their broad role in the

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regulation of plant immunity.In this study, we identified VdSCP41 as a virulence

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effector that suppresses plant immunity induced by PAMPs. VdSCP41 interacts with

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the Arabidopsis CBP60g and SARD1 and modulates their transcription factor activity.

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The contribution of VdSCP41 in V. dahliae virulence is significantly reduced during

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the infection of the cbp60g-1/sard1-1 double mutant. GhCBP60b, the closest protein

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homolog of CBP60g in cotton, is also targeted by VdSCP41 and contributes to cotton

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resistance against V. dahliae. Taken together, our findings revealed that CBP60g,

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SARD1 and GhCBP60b are novel components governing V. dahliae resistance and

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that these proteins are modulated by a secretory effector VdSCP41.

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RESULTS

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VdSCP41 Contributes to V. dahliae Virulence in Arabidopsis and Cotton Plants

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Secretome analyses revealed greater than 700 potential secreted proteins in V. 6

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dahliae (Klosterman et al., 2011). To date, relatively few secreted proteins carrying

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virulence function have been characterized in V. dahliae. We performed a reverse

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genetic screen to identify secreted proteins that are crucial for V. dahliae virulence.

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Using the newly developed USER-ATMT-DS binary vector (Wang et al., 2016), we

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constructed 56 gene deletion mutants in V. dahliae strain 592 (V592) that each targets

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an individual potentially secreted protein. The resulting mutants were subjected to

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virulence assessment in host plants, including upland cotton and the model plant

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Arabidopsis. A mutant carrying a targeted deletion of VdSCP41 (VdΔscp41) was

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isolated (Figure 1A) and shown to display significantly reduced virulence compared

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with WT strain V592.

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VdSCP41 encodes a hypothetical protein in the secretome of the V. dahliae Ls.17

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strain (VdLs.17) (Klosterman et al., 2011), and the expression of this gene is

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significantly up-regulated at 2 days post-inoculation (dpi) in Arabidopsis (Figure

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1-figure supplement 1), suggesting a putative function of VdSCP41 in V. dahliae

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infection. Targeted gene deletion of VdSCP41 in the VdΔscp41 mutant was verified

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by Southern blotting (Figure 1B). The VdΔscp41 mutant displayed much weaker

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disease symptoms than WT V592 in both upland cotton (Gossypium hirsutum) (Figure

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1C) and Arabidopsis (Figure 1D). Disease index analyses indicated significantly

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reduced virulence of the VdΔscp41 mutant compared with that of V592 in both

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upland cotton and Arabidopsis (Figure 1E-F, Figure 1—source data 1). The reduced

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virulence of the VdΔscp41 mutant was restored upon complementation with

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GFP-tagged VdSCP41 (VdΔscp41/VdSCP41-GFP) (Figure 1C-F). Thus, VdSCP41

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functions as a virulence effector that contributes to V. dahliae virulence on host plants.

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VdSCP41 Acts as an Intracellular Effector

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In V. dahliae, some signal peptide-containing SCPs are delivered to the

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septin-organized hyphal neck, which develops from the base of the hyphopodia and

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functions as a fungus-host penetration interface for the dynamic delivery of secretory

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proteins (Zhou et al., 2017). VdSCP41 contains an N-terminal signal peptide

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predicted by SignalP (http://www.cbs.dtu.dk/services/SignalP/) (Figure 2-figure

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supplement 1A). Therefore, we examined the localization of VdSCP41 in V. dahliae.

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GFP-tagged VdSCP41 (VdSCP41-GFP) was found to localize to the base of the

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hyphopodium and showed ring signals surrounding the hyphal neck when the

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Vd∆scp41

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(Vd∆scp41/VdSCP41-GFP)

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hyphopodium induction (Figure 2A). In contrast, VdSCP41 lacking signal peptide

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(DspVdSCP41-GFP) showed diffused signal at the base of the hyphopodium without

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clear ring signals surrounding the hyphal neck. The results demonstrate that

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VdSCP41-GFP was delivered to the penetration interface for secretion.

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Nuclear

mutant

strain

localization

was

complemented cultured

signal

on

a

(NLS)

with cellophane

sequence

VdSCP41-GFP membrane

for

prediction

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(http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) identified a potential

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NLS in VdSCP41 (Figure 2-figure supplement 1A). We therefore took advantage of

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this NLS to examine the putative translocation of V. dahliae-delivered VdSCP41 into 8

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the nucleus of plant cells. The Vd∆scp41/VdSCP41-GFP and GFP-expressing V592

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(V592-GFP) strains were separately inoculated onto onion epidermal cells. While

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GFP fluorescence from the V592-GFP strain was observed in conidial spores,

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VdSCP41-GFP secreted by V. dahliae was capable of translocating into plant cells and

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localizing to the nucleus, in addition to the pericellular space of onion epidermal cells

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(Figure 2-figure supplement 1B). The potential NLS sequence of SCP41 was mutated

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(SCP41-nls-GFP)

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theVdΔscp41/VdSCP41-nls-GFP

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VdSCP41-nls-GFP failed to translocate into the nucleus of onion epidermal cells

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(Figure 2-figure supplement 1B). These results suggested that VdSCP41 delivered by

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V. dahliae translocates into the nucleus of the plant cell, which requires the predicted

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NLS within its sequence.

and

then

complemented strain.

In

into

Vd∆scp41

contrast

to

to

construct

VdSCP41-GFP,

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We next transiently expressed mCherry-tagged VdSCP41 in plants to further

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verify its nuclear localization in plant cells. As the signal peptide located at the N

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terminus may guide secreted proteins into the plant extracellular space in some cases,

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we fused mCherry to VdSCP41 both with and without (DspVdSCP41) the signal

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peptide

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DspVdSCP41-mCherry were individually transiently expressed in either Arabidopsis

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protoplasts or Nicotiana benthamiana (N. b.) leaves. mCherry fluorescence imaging

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revealed that both VdSCP41 and DspVdSCP41 localized to the nucleus in

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Arabidopsis cells (Figure 2B). The protein expression level of VdSCP41-mCherry and

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DspVdSCP41-mCherry was detected by immunoblot (Figure 2-figure supplement 1C).

for

analyses

of

subcellular

localization.

9

VdSCP41-mCherry

and

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Similar nuclear localization was observed for both VdSCP41-mCherry and

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DspVdSCP41-mCherry at the nucleus of N. b. cells (Figure 2-figure supplement 1D).

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These results are consistent with the nuclear localization of the V. dahliae-delivered

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VdSCP41 in onion epidermal cells.

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VdSCP41 Expression in Arabidopsis Inhibits Plant Immunity

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It is believed that the initial function of a fungal effector protein is to suppress

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PTI. To investigate whether VdSCP41 is capable of inhibiting plant immunity when it

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is directly expressed in plants, Arabidopsis transgenic lines expressing DspVdSCP41

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were constructed and assessed for PAMP-induced defense responses. Flg22 is the

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best-characterized PAMP derived from a bacterial pathogen, and it induces the

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expression of PTI -responsive genes in wildtype (WT) Arabidopsis plants. We

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observed

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VdSCP41-expressing lines (Figure 2-figure supplement 1E), suggesting inhibition of

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PTI conferred by VdSCP41.

reduced

induction

of

flg22-induced

ICS1

in

two

independent

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NLP proteins derived from bacterial, oomycete and fungal organisms have

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recently been characterized as PAMPs. A conserved 20-amino acid peptide (nlp20)

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within NLP represents the active immunogenic motif that induces PTI in plants

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(Albert et al., 2015; Böhm et al., 2014; Ottmann et al., 2009). We previously reported

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the immune-inducing activity of VdNLP1 and VdNLP2 derived from V592 in N. b.,

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Arabidopsis, and cotton plants (Zhou et al., 2012). A corresponding peptide located in

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VdNLP2 derived from V592 (nlp20Vd2) was synthesized and shown to cause 10

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up-regulation of cotton PR genes (Du et al., 2017), indicating the immunogenic

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activity of this peptide in cotton. Treatment of Arabidopsis with nlp20Vd2 significantly

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induced ICS1 and FMO1 expression (Figure 2C-D, Figure 2 — source data 1),

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indicating that nlp20Vd2 also exhibits the active immunogenic activity characteristic of

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a PAMP in Arabidopsis. In transgenic lines expressing VdSCP41, but not transgenic

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lines expressing NLS mutated VdSCP41 (DspVdSCP41-nls), suppression of

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nlp20Vd2-induced ICS1 (Figure 2C) and FMO1 (Figure 2D) was observed, suggesting

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that VdSCP41 expression in plants inhibits nlp20Vd2-triggered immunity. ICS1

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encodes the key enzyme controlling SA production and is required for

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pathogen-induced SA accumulation (Wildermuth et al. 2001). We next quantified SA

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production in response to a nonpathogenic bacterial pathogen, Pst DC3000 hrcC-, in

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both WT and DspVdSCP41-expressing plants. In consistent with the suppression of

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PAMP-induced

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accumulated less free SA in response to Pst hrcC- than did WT plants (Figure 2-figure

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supplement 2A).

ICS1

expression,

transgenic

lines

expressing

DspVdSCP41

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Inoculation of WT V. dahliae strain V592 on Arabidopsis induced the expression

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of ICS1 and FMO1 (Figure 2-figure supplement 2B-C). This induced expression was

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further enhanced when the plants were inoculated with the VdDscp41 mutant

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compared with V592 (Figure 2-figure supplement 2B-C), indicating that VdSCP41

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suppresses the induced expression of ICS1 and FMO1 during V. dahliae infection.

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Taken together, these results reveal an inhibitory role of VdSCP41 in modulating

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plant immunity. 11

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Arabidopsis Master Immune Regulators CBP60g and SARD1 are Targeted by

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VdSCP41

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Modulating the activity of plant immune components is a common strategy used

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by effectors to suppress host immunity. To explore the virulence mechanisms

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employed by VdSCP41 in inhibiting plant immunity, we next searched for plant

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components that are targeted by VdSCP41. DspVdSCP41 was fused with a 3×FLAG

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tag and transiently expressed in Arabidopsis protoplasts followed by purification of

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VdSCP41-containing protein complexes. Protein lysates were immuno-precipitated

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using anti-FLAG-conjugated beads and subjected to tandem mass spectrometry. The

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plant CBP CBP60g was identified as a candidate interactor of VdSCP41

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(Supplementary File 1-Table S1).

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CBP60g was then fused with a 3×HA tag and used for reverse

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co-immunoprecipitation (Co-IP) analysis to verify its interaction with VdSCP41.

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FLAG-tagged VdSCP41 was transfected, either alone or together with HA-tagged

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CBP60g, into Arabidopsis protoplasts for transient expression. Anti-HA IP followed

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by an anti-FLAG immunoblot revealed that VdSCP41 was co-purified with CBP60g

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from plant cells (Figure 3-figure supplement 1A). VdSCP41 was further divided into

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an N-terminal portion (VdSCP41N, amino acid 1-213) and a C-terminal portion

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(VdSCP41C, amino acid 163-end),which exhibited an overlap of 50 amino acids, and

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was used to test interactions with CBP60g. Co-IP analysis revealed that VdSCP41C is

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sufficient for interacting with CBP60g (Figure 3A). 12

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Quantitative luciferase complementation imaging assays were performed to

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further verify the interaction between VdSCP41 and CBP60g in N. b. Co-expression

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of the N-terminal region of luciferase (NLuc)-tagged BIK1 and the C-terminal region

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of luciferase (CLuc)-tagged XLG2 driven by the35S promoter was performed as a

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positive interaction control (Liang et al., 2016). The co-expression of NLuc-VdSCP41

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and CLuc-CBP60g driven by the 35S promoter in N. b. resulted in much higher

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luciferase activity than co-expression of NLuc-VdSCP41 and CLuc-XLG2, or

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CLuc-CBP60g and NLuc-BIK1 (Figure 3B-C, Figure 3—source data 1), confirming

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the interaction between VdSCP41 and CBP60g in the plants. The expression levels of

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the NLuc- and CLuc-fusion proteins were further detected via immunoblotting

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(Figure 3-figure supplement 1B).

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The nuclear localization of VdSCP41 prompted us to examine whether CBP60g

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co-localizes with VdSCP41 in the nucleus. GFP-tagged CBP60g mainly localized in

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the nucleus when it was expressed alone in N. b. cells (Figure 3-figure supplement

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1C). GFP-tagged CBP60g was co-expressed with mCherry-tagged VdSCP41 without

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signal peptide (DspVdSCP41-mCherry) in N. b. leaves via Agrobacterium-mediated

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transient expression. An overlay of the results of GFP and mCherry fluorescence

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imaging indicated co-localization of DspVdSCP41 and CBP60g in the nucleus in N. b.

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cells

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DspVdSCP41-mCherry and DspVdSCP41-nls-mCherry was detected by immunoblot

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with the indicated antibodies (Figure 3-figure supplement 1D). In addition to

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co-localization, co-expression of DspVdSCP41-mCherry significantly increased the

(Figure

3D).

The

protein

expression

13

level

of

GFP-CBP60g,

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nuclear accumulation of CBP60g-GFP (Figure 3D), which was not observed when

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DspVdSCP41-nls-mCherry with a mutated NLS was co-expressed with CBP60g-GFP

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(Figure 3D).

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CBP60g was induced by pathogen and PAMP treatments and required for full

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resistance against bacterial pathogens (Wang et al., 2011; Zhang et al., 2010b). A

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closely related protein in the CBP family, SARD1, functions partially redundantly

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with CBP60g in bacterial resistance (Sun et al., 2015; Wang et al., 2011). We also

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detected an interaction between VdSCP41 and SARD1 by Co-IP in Arabidopsis

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protoplasts (Figure 3-figure supplement 2A). VdSCP41 co-localized with SARD1 in

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N. b. leaves and increased its nuclear accumulation (Figure 3-figure supplement 2B),

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indicating similar targeting of SARD1 by VdSCP41. It is unlikely an interaction

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between CBP60g and SARD1 because the luciferase complementation assay did not

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show interaction between CLuc-tagged CBP60g and NLuc-tagged SARD1 (Figure

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3-figure supplement 3). Taken together, the above results demonstrated that both

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CBP60g and SARD1 are targeted by VdSCP41.

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VdSCP41 Interferes with the Transcription Factor Activity of CBP60g

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CBP60g encodes a plant-specific transcription factor that regulates the

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expression of a number of defense-related genes. The fact that CBP60g functions as

305

master transcription regulator (Sun et al., 2015; Wang et al., 2011) prompted us to

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examine whether the targeting of CBP60g by VdSCP41 affects the induction of its

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target genes. Dual reporter analyses revealed that CBP60g expression in Arabidopsis 14

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protoplasts significantly enhanced the expression of ICS1::LUC or FMO1::LUC

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(firefly luciferase) (Figure 4A-B, Figure 4 — source data 1). Co-expression of

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VdSCP41 inhibited CBP60g-induced ICS1::LUC (Figure 4A) and FMO1::LUC

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(Figure 4B) expression, whereas the co-expression of VdSCP41N, which is unable to

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bind CBP60g, was impaired in this inhibition (Figure 4A-B). CBP60g was next

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divided into an N-terminal portion (CBP60gN, amino acid 1-361) containing its

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DNA-binding domain and a C-terminal portion (CBP60gC, amino acid 211-end)

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lacking the functional DNA-binding domain (Zhang et al., 2010b), which exhibited an

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overlap of 150 amino acids. CBP60gN and CBP60gC were then used to test

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interactions with VdSCP41. Co-IP analysis showed that VdSCP41 binds to CBP60gC,

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but not CBP60gN (Figure 4C). The results indicated that VdSCP41 binds the

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C-terminal portion of CBP60g to interfere with its activity.

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To test whether VdSCP41 targeting directly affects the DNA-binding activity of

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CBP60g, recombinant GST-tagged CBP60g and His-tagged VdSCP41C were purified

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and used for electrophoretic mobility shift assays (EMSAs). GST-CBP60g showed a

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specific binding to a 60-bp DNA fragment (ICS1 promoter probe) within the ICS1

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promoter, which is reduced by the addition of unlabelled probe, as previously reported

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(Zhang et al. 2010) (Figure 4D). The preincubation of VdSCP41C with CBP60g

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significantly reduced the DNA-binding activity of CBP60g, whereas a soluble

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fragment of His-tagged VdSCP41 which does not contain its C-terminal portion

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(VdSCP41100-163) did not (Figure 4D). Another His-tagged V. dahliae protein

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(VDAG_01962) also did not affect the DNA-binding activity of CBP60g (Figure 4D). 15

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Coexpression of VdSCP41 did not lead to cleavage or mobility shift of CBP60g

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(Figure 4-figure supplement 1), suggesting that VdSCP41 is unlikely to act as a

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protease to target CBP60g. The results proved that binding of VdSCP41C to CBP60g

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directly inhibits the DNA-binding activity of CBP60g.

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CBP60gC Harbors a Transcription Activation Domain that is Required for

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Interacting with VdSCP41

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The binding of VdSCP41 to the C-terminal portion of CBP60g prompted us to

338

test the role of CBP60gC in CBP60g-mediated gene activation. Compared to the full

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induction of ICS1::LUC or FMO1::LUC by CBP60g (Figure 5A, Figure 5—source

340

data 1), the deletion of the C-terminal portionDC-CBP60g) dramatically

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compromised its activity to induce both ICS1::LUC and FMO1::LUC (Figure 5A),

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indicating that CBP60gC is required for CBP60g-mediated gene activation. The

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results suggest that putative transcription activator activity may be harbored within

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the CBP60gC. The basic helix-loop-helix (bHLH) transcription factor MYC2 directly

345

binds to the G-box-like (CANNTG) element (Dombrecht et al., 2007; Godoy et al.,

346

2011; Lian et al., 2017) via its bHLH domain to regulate the expression of its target

347

genes, such as the TERPENE SYNTHASE gene 10 (TPS10) (Li et al., 2014b). We

348

therefore took advantage of bHLH-mediated binding to the TPS10 promoter to

349

examine the putative transcription activator activity of CBP60gC. A fragment within

350

the C-terminal portion of CBP60g (amino acid 211-440) activated TPS10::LUC

351

reporter when it was fused with the bHLH domain of MYC2 (bHLH-CBP60g211-440) 16

352

(Figure 5B, Figure 5—source data 1), compared with bHLHMYC2 alone, indicating that

353

the CBP60g211-440 harbors transcription activator activity. Moreover, Co-IP analysis

354

showed that CBP60g211-end, but not CBP60g441-end, co-purified with VdSCP41,

355

indicating the requirement of CBP60g211-440 for binding to VdSCP41 (Figure 5C).

356

Thus, CBP60gC harbors a transcription activation domain, CBP60g211-440, that is

357

required for VdSCP41 targeting.

358

The DC-CBP60g was further co-transfected with CBP60g, together with

359

ICS1::LUC or FMO1::LUC. Dual reporter analyses indicated that co-expression of

360

DC-CBP60g significantly suppressed CBP60g-induced ICS1::LUC and FMO1::LUC

361

(Figure 5A), suggesting a dominant negative effect of DC-CBP60g on the activity of

362

CBP60g.

363 364

CBP60g and SARD1 Contribute to Arabidopsis Resistance against V. dahliae

365

SARD1 functions both redundantly and differentially with CBP60g (Sun et al.,

366

2015; Wang et al., 2011). The finding that VdSCP41 targeted both CBP60g and

367

SARD1 prompted us to assess the roles of CBP60g and SARD1 in resistance to V.

368

dahliae. The WT and cbp60g-1/sard1-1 double mutant plants were inoculated with

369

V592 for the assessment of disease symptoms. As shown in Figure 6, the

370

cbp60g-1/sard1-1 double mutant displayed compromised resistance compared with

371

the WT plants, demonstrating a contribution of CBP60g and SARD1 to V. dahliae

372

resistance.

373

We next investigated the requirement of CBP60g and SARD1 for 17

374

VdSCP41-mediated virulence. The WT and cbp60g-1/sard1-1 double mutant plants

375

were inoculated with the VdDscp41 mutant. The VdDscp41 mutant displayed reduced

376

virulence on the WT plants compared with V592. The reduced virulence arising from

377

VdSCP41 deletion was partially impaired in the cbp60g-1/sard1-1 double mutant

378

plants compared with than in the WT plants (Figure 6, Figure 6—source data 1). The

379

results indicated that both CBP60g and SARD1 are required for full virulence

380

conferred by VdSCP41. However, we still observed reduced virulence of the

381

VdDscp41 mutant compared with V592 on the cbp60g-1/sard1-1 double mutant plants,

382

suggesting the existence of additional targets for VdSCP41 during V. dahliae

383

infection.

384 385

VdSCP41 Interacts with and Co-localizes with GhCBP60b

386

As in Arabidopsis, we observed similar compromised virulence of VdΔscp41

387

mutant in upland cotton (Figure 1E) compared with V592. The results prompted us to

388

test the putative VdSCP41 targeting of GhCBP60b, the closest protein homolog of

389

CBP60g and SARD1 in cotton. Co-IP analysis revealed an interaction between

390

VdSCP41 and GhCBP60b (Figure 7A). To examine the subcellular localization of

391

GhCBP60b, GhCBP60b-GFP was constructed for transient expression in N. b. leaves.

392

GhCBP60b-GFP localized to the nucleus of N. b. cells, and co-expression of

393

GhCBP60b-GFP with VdSCP41-mCherry revealed co-localization of VdSCP41 and

394

GhCBP60b in the nucleus of N. b. cells (Figure 7B), suggesting conserved targeting

395

of Arabidopsis CBP60g and SARD1 and cotton GhCBP60b by VdSCP41.

396 18

397

GhCBP60b is Required for Cotton Resistance against V. dahliae

398

To further examine whether GhCBP60b functions in resistance to V. dahliae in

399

cotton, we generated a virus-induced gene silencing (VIGS) vector (Liu et al., 2002)

400

targeting GhCBP60b (pTRV2-GhCBP60b). Cotton plants were infiltrated with

401

pTRV1 together with pTRV2 or pTRV2-GhCBP60b and further inoculated with V.

402

dahliae. Compared with pTRV2, pTRV2-GhCBP60b-infiltrated cotton plants

403

exhibited higher ratios of wilting (Figure 7C, Figure 7—source data 1) and more

404

severe disease symptoms (Figure 7D-E, Figure 7—source data 1), indicating a role for

405

GhCBP60b in cotton resistance to V. dahliae. The reduced expression of GhCBP60b

406

in pTRV2-GhCBP60b-infiltrated plants was verified via RT-PCR (Figure 7F, Figure 7

407

—source data 1). The results support the targeting of GhCBP60b by VdSCP41 for

408

virulence during V. dahliae infection in cotton.

409 410

DISCUSSION

411

In this study, we identified VdSCP41 as an intracellular effector that is crucial

412

for V. dahliae virulence. VdSCP41 targets Arabidopsis CBP60g and SARD1 to

413

interfere with the induction of their target genes. CBP60g and SARD1 are required for

414

Arabidopsis resistance to V. dahliae and VdSCP41-mediated virulence. Silencing of

415

GhCBP60b compromised cotton resistance to V. dahliae. The results revealed a

416

conserved and important role for Arabidopsis CBP60g and SARD1 and cotton

417

GhCBP60b, which are modulated by an intracellular effector, in plant resistance

418

against V. dahliae. 19

419 420

VdSCP41 is Secreted by V. dahliae and Translocates into Plant Cells

421

To actively suppress plant defense or modulate host physiology to benefit

422

pathogenic fitness, oomycete and fungal pathogens deliver hundreds of effectors

423

through specialized intracellular fungal structures, such as haustoria and infection

424

hyphaea, into the host apoplastic space or directly into plant cells (Lo Presti et al.,

425

2015). Although haustoria have not been observed during infection, V. dahliae

426

develops a penetration peg from a hyphopodium when infecting plant roots (Zhao et

427

al., 2016). During V. dahliae infection, the penetration peg developed from the

428

hyphopodium further develops a hyphal neck and forms a septin ring that partitions

429

the hyphopodium and invasive hyphae. This septin-organized apparatus functions as a

430

fungus-host interface for the dynamic delivery of secretory proteins, such as SCPs

431

(Zhao et al., 2016; Zhou et al., 2017).

432

To date, relatively few V. dahliae-secreted effectors have been characterized as

433

functioning inside host cells to modulate host immunity. Here, we provided evidence

434

of the secretion and translocation of VdSCP41 secreted by V. dahliae into plant cells.

435

VdSCP41 contains a signal peptide and localizes to the base of the hyphopodium to

436

form a septin-like ring during infection (Figure 2A), indicating that it is secreted via

437

the septin-organized apparatus at the fungus-host interface. The localization of

438

VdSCP41 delivered by V. dahliae at the nucleus of onion epidermal cells (Figure

439

2-figure supplement 1B) suggested the translocation of VdSCP41 into plant cells. A

440

few conserved motifs have been indicated to serve as signals for the uptake of fungal 20

441

or oomycete effectors into plant cells. For example, a few effectors secreted by cereal

442

powdery mildew and rust pathogens possess a Y/F/WxC motif that serves as a signal

443

for translocation into the plant cell (Godfrey et al., 2010; Spanu et al., 2010). A set of

444

oomycete effectors contain a RXLR-dEER motif that appears to assist in targeting

445

effectors into plant cells (Dou et al., 2008; Whisson et al., 2007). Another set of

446

oomycete effectors possess a FLAK motif for translocation (Schornack et al., 2010).

447

However, VdSCP41 lacks any of the above conserved motifs, and the mechanisms by

448

which assist the uptake of VdSCP41 into plant cells remain unclear.

449

VdSCP41 Targets CBP60g and SARD1 and Interferes with Plant Defense

450

CBP60g and SARD1 are master regulators in immunity. ChIP-seq analyses

451

allowed the identification of a large number of targets genes for CBP60g and SARD1

452

and support a model in which CBP60g and SARD1 accumulate in the plant nucleus

453

and act as master regulators, to activate both positive and negative immune regulators

454

(Sun et al., 2015; Wang et al., 2009; Zhang et al., 2010b). Genetic evidence revealed

455

that CBP60g and SARD1 are positive immune regulators required for immune

456

responses and bacterial resistance (Wang et al., 2009; Zhang et al., 2010b). We

457

demonstrated that VdSCP41 targets CBP60g and SARD1 and interferes with their

458

activity, thus supporting the virulence function of VdSCP41 for immune suppression.

459

Consistent with the inhibition of CBP60g and SARD1 activity, we observed less

460

pathogen-induced SA accumulation in transgenic lines expressing VdSCP41 than in

461

WT plants (Figure 2-figure supplement 2A). As CBP60g and SARD1 also directly

462

bind to the promoters of both ALD1 and SARD4 (Sun et al., 2018), two major genes 21

463

encoding pipecolic acid (Pip) biosynthesis enzymes, to regulate Pip biosynthesis, a

464

potential contribution of Pip to V. dahliae resistance will be of interest for further

465

investigation.

466

We showed that VdSCP41 binds the C-terminal portion of CBP60g to interfere

467

with its transcription factor activity (Figure 4A-B). Coexpression of VdSCP41 did not

468

lead to cleavage or mobility shift of CBP60g (Figure 4-figure supplement 1),

469

suggesting that VdSCP41 is unlikely to act as a protease to target CBP60g. In addition,

470

CBP60g211-440, a domain within the C-terminal portion of CBP60g, harbors

471

transcription activator activity (Figure 5B) and is required for interacting with

472

VdSCP41 (Figure 5C). It is likely that CBP60gC functions to promote transcriptional

473

activation via recruiting additional activators, and binding of VdSCP41 interrupts the

474

activity of this domain or the recruitment of associated activators via this domain. The

475

dominant negative function of DC-CBP60g (Figure 5A) supports a dominant negative

476

effect on CBP60g activity arising from increased nuclear accumulation of

477

VdSCP41-impaired CBP60g. Thus, VdSCP41 mediated over-accumulation of

478

CBP60g provides an additional strategy to further interfere with its transcription

479

factor activity. The results suggest a novel virulence strategy in which a pathogenic

480

effector directly targets host transcription factors to interfere with their activity and

481

modulate plant immunity. However, it is equally possible that the increased nuclear

482

accumulation of CBP60g results from the feed-back regulation as a result of impaired

483

CBP60g function.

484

VdSCP41 Functions to Suppress Immunity 22

485

CBP60g and SARD1 are key components of the SA signaling pathway, which

486

serves as an attractive target for bacterial and fungal effectors (DebRoy et al., 2004;

487

Djamei et al., 2011; Nomura et al., 2011). Targeting of CBP60g and SARD1 by

488

VdSCP41 may therefore interfere with plant resistance to V. dahliae via the

489

manipulation of SA signaling. On the other hand, ChIP-seq analyses identified a

490

number of SA-independent regulators that are directly targeted by CBP60g and

491

SARD1 (Sun et al., 2015), revealing broader and SA-independent functions of

492

CBP60g and SARD1 in immunity. It is likely that signaling pathways other than SA

493

are also targeted by VdSCP41 through CBP60g and SARD1 to interfere with plant

494

immunity. Consistent with this assumption, we showed that nlp20Vd2 functions to

495

trigger the induction of both SA-dependent and SA-independent defense genes during

496

V. dahliae infection (Figure 2C-D). Furthermore, VdSCP41 expression suppressed the

497

induction of both SA-dependent ICS1 and SA-independent FMO1 by nlp20Vd2 (Figure

498

2C-D). Modulation of CBP60g and SARD1 may result in interference with their

499

function as master transcription factors in PTI and in other defense pathways.

500

Taken together, our results support a model in which PAMPs from V. dahliae,

501

such as NLPs and chitins, are recognized by plants to induce the expression of

502

CBP60g and SARD1, which subsequently regulate the expression of a number of

503

immune regulators to defend against pathogen infection (Figure 8A). VdSCP41

504

secreted by V. dahliae functions as an intracellular effector that targets the

505

transcription activator domain of CBPs, interrupting the function of this domain, to

506

interfere with their transcription factor activity, and thus modulates both 23

507

SA-dependent and SA-independent regulators to inhibit plant immunity against V.

508

dahliae (Figure 8B).

509

24

510

MATERIAL AND METHODS Key Resources Table Reagent type (species) or

Designation

resource gene (Verticillium dahliae)

VdSCP41

gene (Gossypium hirsutum)

GhCBP60b

strain, strain background (Verticillium dahliae) other (cotton plant) antibody antibody antibody antibody

Source or reference PMID:21829347

PMID:21151869

Xinluzao No. 16

PMID:28282450

monoclonal) anti-HA (mouse monoclonal) anti-GFP (mouse monoclonal) anti-CLuc (mouse monoclonal)

Additional information

NCBI Gene ID:20709665 NCBI Gene ID:107899061

V592

anti-FALG (mouse

Identifiers

Sigma

RRID:AB_259529

1:10000 dilution

Roche

RRID:AB_514506

1:5000 dilution

Roche

RRID:AB_390913

1:3000 dilution

Sigma

RRID:AB_439707

1:3000 dilution

antibody

anti-mCherry

Easybio

1:3000 dilution

peptide

nlp20Vd2

PMID:28755291

recombinant DNA reagent

pTRV1 (VIGS vector)

PMID:12220268

recombinant DNA reagent

pTRV2 (VIGS vector)

PMID:12220268

511 25

512

Fungal strains, plant materials and antibodies.

513

The Verticillium dahliae strain V592 (Gao et al., 2010) was used in this study.

514

Verticillium dahliae strains were grown on potato dextrose agar (PDA) medium at

515

25°C in the dark. To collect conidia, the mycelial plugs were cultured in potato

516

dextrose broth (PDB) liquid medium with shaking at 200 rpm at 25°C for 3-5 days.

517

Cotton plants (`Xinluzao No. 16') were used for virulence assessment in this study

518

(Zhou et al., 2017). Arabidopsis thaliana plants used in this study includes Col-0

519

(wild-type), the cbp60g-1/sard1-1 mutant (Zhang et al., 2010b). VdSCP41 was

520

amplified from the V592 cDNA and cloned into pCambia1300-35S-FLAG (Zhang et

521

al., 2010a) to construct Arabidopsis transgenic lines expressing VdSCP41. Antibodies

522

used

523

(RRID:AB_514506), anti-CLuc (RRID:AB_439707), anti-GFP (RRID:AB_390913),

524

and anti-mCherry (Easybio, BE2026).

525

Generation of deletion and complementation fungal strains

526

The upstream and downstream flanking sequences were PCR amplified from V592

527

genomic DNA and cloned into pGKO-HPT vector (Wang et al., 2016). The resulting

528

construct was transformed into Agrobacterium tumefaciens EHA105, and used for

529

Agrobacterium tumefaciens-mediated transformation (ATMT) to generate the

530

VdΔscp41 mutant strain (Wang et al., 2016). Genomic region, including 1.5 kb

531

upstream from the start codon, of VdSCP41 was amplified and cloned into

532

pNat-Tef-TrpC vector (Zhou et al., 2017) to generate construct for complementation.

533

The resulting construct was transformed into Agrobacterium tumefaciens EHA105,

in

this

study

includes

anti-FLAG

26

(RRID:AB_259529),

anti-HA

534

and used for ATMT to generate VdΔscp41/VdSCP41 strain. Primers used in this

535

study are listed in Supplementary File 2.

536

Protein complex purification and mass spectrum analysis

537

Arabidopsis protoplasts isolated from 10 gram leaves were transfected with

538

DspVdSCP41-FLAG, DspVdSCP45-FLAG, or empty vector for protein expression.

539

Transfected protoplasts were collected and total protein was extracted with extraction

540

buffer containing 50mM HEPES, pH7.5, 150 mM KCl, 1 mM EDTA, 1 mM DTT,

541

0.2% Triton X-100, and 1×proteinase inhibitor cocktail. Total protein was incubated

542

with 50μl anti-FLAG agarose beads (sigma) for 12 hours at 4°C. The

543

immunocomplex was washed three times using the above buffer and eluted with

544

100μl 1μg/μl 3×FLAG peptide. The eluted proteins were run 10 mm into the

545

separating gel and stained with Proteo Silver stain kit (Sigma). Total protein was

546

destained and digested in-gel with sequencing grade trypsin (10 ng/mL trypsin, 50

547

mM ammonium bicarbonate [pH 8.0]) overnight. Peptides were sequentially extracted

548

with 5% formic acid/50% acetonitrile and 0.1% formic acid/75% acetonitrile and

549

concentrated to 20 ml. The extracted peptides were separated by an analytical capillary

550

column packed with 5 mm spherical C18 reversed-phase material. The eluted peptides

551

were sprayed into a LTQ mass spectrometer (Thermo Fisher Scientific) equipped with

552

a nano-ESI ion source. The mass spectrometer was operated in data-dependent mode

553

with one MS scan followed by five MS/MS scans for each cycle. Database searches

554

were performed on an in-house Mascot server (Matrix Science Ltd., London, UK)

555

against IPI (International Protein Index) Arabidopsis protein database. 27

556

Co-immunoprecipitation assay in protoplasts

557

Five-week-old

558

pUC-35S-VdSCP41-FLAG, or its variants construct, was co-transfected with

559

pUC-35S-CBP60g-HA,

560

pUC-35S-GhCBP60b-HA, into Arabidopsis protoplasts. Total protein was extracted

561

with extraction buffer. For anti-HA IP, total protein was incubated with 2 μg of

562

anti-HA antibody together with protein A agarose at 4°C for 4 hours. The agarose

563

were collected and boiled for 5 minutes with 1× protein loading buffer.

564

Immunoprecipitates were separated by 10% SDS-PAGE and the presence of

565

VdSCP41-FLAG or its variants, CBP60g-HA or its variants, SARD1-HA, or

566

GhCBP60b-HA was detected by anti-FLAG or anti-HA immunoblot respectively.

567

SA Measurement

568

SA was extracted and measured following the method described previously (Sun et al.,

569

2015). Around 0.3 gram leaf tissue collected from 4-week-old plants was ground into

570

powder in liquid nitrogen. Plant leaves were infiltrated with or without Pst DC3000

571

hrcC- (OD600=0.1) 12 hours before sample collection. Three samples for each

572

treatment were analysed. The samples were extracted with 0.8 mL 90% methanol and

573

sonicated for 15 min, and the supernatant was transferred into a new tube. The pellet

574

was re-extracted with 0.5 mL of 100% methanol, the supernatant was combined with

575

the first-step supernatant and dried by vacuum. The pellet was resuspended in 500 μL

576

0.1 M sodium acetate (pH 5.5) in 10% methanol. An equal volume of 10% TCA was

577

added and the samples were vortexed, sonicated for 5min. After centrifuged, the

Arabidopsis

or

plants

its

were

variants

28

used

construct,

for

protoplast

isolation.

pUC-35S-SARD1-HA,

or

578

supernatant was extracted three times with 0.5 ml of extraction buffer

579

(ethylacetate/cyclopentane/isopropanol:100/99/1 by volume). After spinning, the

580

organic phases were collected and dried by vacuum. The samples were then dissolved

581

in 250 μL 100% methanol and filtered through a 0.22 μm filter. The samples were

582

then assayed by HPLC-MS/MS analysis on AB SCIEX QTRAP 4500 system (AB

583

SCIEX, Foster, CA, USA).

584

Luciferase complementation imaging assay

585

Agrobacterium tumefaciens GV3101 strain carrying CLuc or NLuc tagged consctruct

586

were infiltrated into leaves of 4-week-old Nicotiana benthamiana. LUC activity in

587

leaves was examined 2 days post infiltration. N. b. leaves were treated with 1 mM

588

luciferin and kept in dark for 5 min to quench the fluorescence. LUC image was

589

captured by CCD imaging apparatus and the quantitative LUC activity was

590

determined by microplate luminometer. Expression of CLuc-tagged proteins or

591

NLuc-tagged proteins was detected by anti-CLuc or anti-HA western blot,

592

respectively.

593

Electrophoretic mobility shift assays (EMSAs)

594

The full-length CBP60g was cloned into the pGEX-6p-1 vector. VdSCP41C,

595

VdSCP41100-163 or VDAG_01962 was cloned into the pET-28a vector. The resulting

596

constructs were transformed into in E. coli BL21 (DE3) competent cells. BL21 (DE3)

597

strains containing the expression vectors were cultured and induced by IPTG at 16°C

598

for 16 hours. GST-tagged full-length CBP60g, His-tagged VdSCP41C or

599

VdSCP41100-163, and His-tagged VDAG_01962 recombinant protein were affinity 29

600

purified and used for EMSAs. A 60-bp probe within the DNA fragment 7 (Zhang et

601

al., 2010) in the ICS1 promoter were labeled with [γ-32P]ATP using T4 polynucleotide

602

kinase. Binding reactions were carried out in a 20μl volume of reaction buffer [10

603

mM Tris-HCl, pH 7.5, 50 mM KCl, 1mM DTT, 1 μl 50 ng/μl poly(dI-dC)] for 30 min

604

at room temperature. Labeled DNA probe (2 fmol) was incubated with 4 μg

605

GST-CBP60g. 150× unlabeled DNA probe was used for competition. 1.5 μg

606

His-tagged VdSCP41C163-end, VdSCP41100-163, or VDAG_01962 was preincubated

607

with GST-CBP60g for 30 min at room temperature before DNA binding. The reaction

608

was stopped by adding DNA loading buffer and the samples were separated by a 5%

609

native PAGE gel. After electrophoresis, the gel was autoradiographed.

610

RNA isolation and qRT-PCR

611

Total RNA was isolated with the TRIzol reagent (Invitrogen) and used for cDNA

612

synthesis with SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen)

613

following the instructions provided by the manufacturer. The quantitative PCR were

614

performed with SYBR Premix Ex Taq kit (TaKaRa) following standard protocols.

615

Arabidopsis col-0 or transgenic plants were treated with H2O, 1μM flg22 or nlp20Vd2

616

(Du et al., 2017) as indicated for 3 hours. RNA was isolated and used for RT-PCR

617

analysis for the expression of ICS1 and FMO1. AtTUB4 was used as internal control.

618

To detect gene expression in cotton plants, leaves from cotton plants were collected

619

14 days post Agrobacterium infiltration. Quantitative RT-PCR was performed as

620

described above. G. hirsutum HISTONE3 was used as internal control. For RT-PCR

621

analyses of VdSCP41, V592 or VdΔscp41 strain was incubated with the roots of 30

622

7-day-old Arabidopsis col-0 plants for 2 days. Conidia were collected for RNA

623

isolation and RT-PCR analysis for the expression of VdSCP41. For RT-PCR analyses

624

of V. dahliae-infected Arabidopsis plants, Arabidopsis plants infected with or without

625

V592 or VdΔscp41 strain were collected for RNA isolation and RT-PCR analysis for

626

the expression of ICS1 and FMO1. VdELF1 was used as internal control for VdSCP41.

627

AtTUB4 was used as internal control for ICS1 and FMO1.

628

Fluorescence microscopy

629

Agrobacterium tumefaciens EHA105 strain carrying pCambia1300-35S-CBP60g-GFP,

630

pCambia1300-35S-SARD1-GFP,

631

infiltrated alone, or together with Agrobacterium tumefaciens EHA105 strain carrying

632

pCambia1300-35S-VdSCP41-mCherry (or its variants mutants) into leaves of

633

4-week-old N. b. GFP and mCherry fluorescence were observed with Leica SP8

634

confocal microscopy 3 days post infiltration. The intensity of fluorescent signals was

635

determined by Image J software. For fluorescence microscopy in Arabidopsis,

636

Arabidopsis protoplasts were transfected with 35S-CBP60g-GFP alone, or together

637

with 35S-VdSCP41-mCherry (or its variants mutants). The protoplasts were incubated

638

overnight under faint light before GFP and mCherry fluorescence were observed. To

639

examine the subcellular localization of VdSCP41, conidia were cultured on

640

cellophane and incubated for 3-9 days before microscopy observation. The cellophane

641

with mycelium were collected and observed as described (Zhou et al., 2017). The

642

plasma membrane of the fungi was stained with FM4-64 (red).

643

Reporter Assay in Arabidopsis Protoplast

or

pCambia1300-35S-GhCBP60b-GFP

31

was

644

Arabidopsis protoplasts were co-transfected with ICS1::LUC or FMO1::LUC, and

645

35S::RLUC (Renilla luciferase), or together with VdSCP41, CBP60g, or their variants

646

as indicated. 12 hours after transfection, protein of transfected protoplasts was

647

isolated, and LUC activity was determined by using Dual-Luciferase® Reporter

648

system (Promega) following manufacture’s instruction.

649

Virus-induced gene silencing in cotton plants

650

The VIGS was performed as described (Gao and Shan, 2013). Cotton plants were

651

grown at 23-25°C in the growth room until two cotyledons have emerged. A 465 bp

652

fragment of GhCBP60b cDNA was PCR amplified from G. hirsutum and cloned into

653

pTRV2 plasmid (Liu et al., 2002). Agrobacterium strain carrying pTRV1 together

654

with Agrobacterium strain carrying pTRV2 or pTRV2-GhCBP60b, as indicated, was

655

infiltrated into the cotyledons of the cotton plants. The cotton plants were root-dip

656

inoculated by V. dahliae V592 2 weeks post Agrobacterium infiltration.

657

Infection assay

658

Cotton or Arabidopsis plants were infected by the root-dip inoculation method (Gao et

659

al., 2010). A conidial suspension of 107/ml from the indicated stains was used as the

660

inoculums. The disease grade was classified as follows: Grade 0 (no symptoms), 1

661

(0-25% wilted leaves), 2 (25-50%), 3 (50-75%) and 4 (75-100%). The disease index

662

was calculated as 100 × [sum (number of plants × disease grade)] / [(total number of

663

plants) × (maximal disease grade)] (Xu et al., 2014). The onion epidermis infection

664

assay was performed as described (Zhang et al., 2017). A conidial suspension of

665

107/ml from V592-GFP, VdΔscp41/SCP41-GFP or VdΔscp41/SCP41-nls-GFP strain 32

666

was inoculated onto the inner layer of onion epidermal cells and incubated on 1%

667

water agar plates for 3-5 days before confocal imaging.

668 669

ACKNOWLEDGMENTS

670

The authors are grateful to Prof. Yuelin Zhang for providing Arabidopsis

671

cbp60g-1/sard1-1 mutant, to Prof. Yule Liu for providing the pTRV1 and pTRV2

672

plasmids, to Prof. Jian Ye for providing the TPS10::LUC plasmid, to Yao Wu for help

673

in SA measurement. This work was supported by grants from the Strategic Priority

674

Research Program of the Chinese Academy of Sciences (XDB11020600) to J. Z., the

675

National Natural Science Foundation of China (31730078) to H-S.G., the Strategic

676

Priority Research Program of the Chinese Academy of Sciences (XDB11040500) to

677

H-S.G., the Youth Innovation Promotion Association of the Chinese Academy of

678

Sciences, and the National Natural Science Foundation of China (31571968,

679

31501593), and the State Key Laboratory of Plant Genomics, Chinese Academy of

680

Sciences.

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REFERENCES

683

Albert, I., Böhm, H., Albert, M., Feiler, C.E., Imkampe, J., Wallmeroth, N., Brancato,

684

C.,

685

RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity. Nat Plants 1,

686

15140.

687

Böhm, H., Albert, I., Oome, S., Raaymakers, T.M., Van den Ackerveken, G., and

Raaymakers,

T.M.,

Oome,

S.,

Zhang,

33

H.Q.,

et

al.

(2015).

An

688

Nurnberger, T. (2014). A conserved peptide pattern from a widespread microbial

689

virulence factor triggers pattern-induced immunity in Arabidopsis. PLoS Pathog 10,

690

e1004491.

691

Boller, T., and He, S.Y. (2009). Innate immunity in plants: an arms race between

692

pattern recognition receptors in plants and effectors in microbial pathogens. Science

693

324, 742-744.

694

Bouche, N., Yellin, A., Snedden, W.A., and Fromm, H. (2005). Plant-specific

695

calmodulin-binding proteins. Annu Rev Plant Biol 56, 435-466.

696

Cui, H.T., Xiang, T.T., and Zhou, J.M. (2009). Plant immunity: a lesson from

697

pathogenic bacterial effector proteins. Cell Microbiol 11, 1453-1461.

698

de Jonge, R., van Esse, H.P., Maruthachalam, K., Bolton, M.D., Santhanam, P., Saber,

699

M.K., Zhang, Z., Usami, T., Lievens, B., Subbarao, K.V., et al. (2012). Tomato

700

immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by

701

genome and RNA sequencing. Proc Natl Acad Sci USA 109, 5110-5115.

702

DebRoy, S., Thilmony, R., Kwack, Y.B., Nomura, K., and He, S.Y. (2004). A family

703

of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and

704

promotes disease necrosis in plants. Proc Natl Acad Sci USA 101, 9927-9932.

705

Djamei, A., Schipper, K., Rabe, F., Ghosh, A., Vincon, V., Kahnt, J., Osorio, S., Tohge,

706

T., Fernie, A.R., Feussner, I., et al. (2011). Metabolic priming by a secreted fungal

707

effector. Nature 478, 395-398.

708

Dombrecht, B., Xue, G.P., Sprague, S.J., Kirkegaard, J.A., Ross, J.J., Reid, J.B., Fitt,

709

G.P., Sewelam, N., Schenk, P.M., Manners, J.M., et al. (2007). MYC2 differentially 34

710

modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19,

711

2225-2245.

712

Dong, X. (2004). NPR1, all things considered. Curr Opin Plant Biol 7, 547-552.

713

Dou, D.L., Kale, S.D., Wang, X., Jiang, R.H.Y., Bruce, N.A., Arredondo, F.D., Zhang,

714

X.M., and Tyler, B.M. (2008). RXLR-mediated entry of Phytophthora sojae effector

715

Avr1b into soybean cells does not require pathogen-encoded machinery. Plant Cell 20,

716

1930-1947.

717

Dou, D.L., and Zhou, J.M. (2012). Phytopathogen effectors subverting host immunity:

718

different foes, similar battleground. Cell Host Microbe 12, 484-495.

719

Du, X., Wang, S., Gao, F., Zhang, L., Zhao, J.H., Guo, H.S., and Hua, C. (2017).

720

Expression of pathogenesis-related genes in cotton roots in response to Verticillium

721

dahliae PAMP molecules. Sci China Life Sci 60, 852-860.

722

Fradin, E.F., Abd-El-Haliem, A., Masini, L., van den Berg, G.C.M., Joosten, M.H.A.J.,

723

and Thomma, B.P.H.J. (2011). Interfamily transfer of tomato Ve1 mediates

724

Verticillium resistance in Arabidopsis. Plant Physiol 156, 2255-2265.

725

Fradin, E.F., and Thomma, B.P.H.J. (2006). Physiology and molecular aspects of

726

Verticillium wilt diseases caused by V. dahliae and V. albo-atrum. Mol Plant Pathol 7,

727

71-86.

728

Fradin, E.F., Zhang, Z., Ayala, J.C.J., Castroverde, C.D.M., Nazar, R.N., Robb, J., Liu,

729

C.M., and Thomma, B.P.H.J. (2009). Genetic dissection of Verticillium wilt resistance

730

mediated by tomato Ve1. Plant Physiol 150, 320-332.

731

Gao, F., Zhou, B.J., Li, G.Y., Jia, P.S., Li, H., Zhao, Y.L., Zhao, P., Xia, G.X., and Guo, 35

732

H.S. (2010). A glutamic acid-rich protein identified in Verticillium dahliae from an

733

insertional mutagenesis affects microsclerotial formation and pathogenicity. Plos One

734

5, e15319.

735

Gao, W., Long, L., Zhu, L.F., Xu, L., Gao, W.H., Sun, L.Q., Liu, L.L., and Zhang, X.L.

736

(2013a). Proteomic and virus-induced gene silencing (vigs) analyses reveal that

737

gossypol, brassinosteroids, and jasmonic acid contribute to the resistance of cotton to

738

Verticillium dahliae. Mol Cell Proteomics 12, 3690-3703.

739

Gao, X., Li, F., Li, M., Kianinejad, A.S., Dever, J.K., Wheeler, T.A., Li, Z., He, P., and

740

Shan, L. (2013b). Cotton GhBAK1 mediates Verticillium wilt resistance and cell death.

741

J Integr Plant Biol 55, 586-596.

742

Gao, X., and Shan, L. (2013). Functional genomic analysis of cotton genes with

743

agrobacterium-mediated virus-induced gene silencing. Methods Mol Biol 975,

744

157-165.

745

Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., and Shan, L. (2011). Silencing

746

GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant J 66,

747

293-305.

748

Godfrey, D., Bohlenius, H., Pedersen, C., Zhang, Z.G., Emmersen, J., and

749

Thordal-Christensen, H. (2010). Powdery mildew fungal effector candidates share

750

N-terminal Y/F/WxC-motif. Bmc Genomics 11, 317.

751

Godoy, M., Franco-Zorrilla, J.M., Perez-Perez, J., Oliveros, J.C., Lorenzo, O., and

752

Solano, R. (2011). Improved protein-binding microarrays for the identification of

753

DNA-binding specificities of transcription factors. Plant J 66, 700-711. 36

754

Gui, Y.J., Chen, J.Y., Zhang, D.D., Li, N.Y., Li, T.G., Zhang, W.Q., Wang, X.Y., Short,

755

D.P.G., Li, L., Guo, W., et al. (2017). Verticillium dahliae manipulates plant immunity

756

by glycoside hydrolase 12 proteins in conjunction with carbohydrate-binding module

757

1. Environ Microbiol 19, 1914-1932.

758

Klimes, A., and Dobinson, K.F. (2006). A hydrophobin gene, VDH1, is involved in

759

microsclerotial development and spore viability in the plant pathogen Verticillium

760

dahliae. Fungal Genet Biol 43, 283-294.

761

Klosterman, S.J., Subbarao, K.V., Kang, S., Veronese, P., Gold, S.E., Thomma, B.P.,

762

Chen, Z., Henrissat, B., Lee, Y.H., Park, J., et al. (2011). Comparative genomics

763

yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog 7,

764

e1002137.

765

Li, C., He, X., Luo, X.Y., Xu, L., Liu, L.L., Min, L., Jin, L., Zhu, L.F., and Zhang,

766

X.L. (2014a). Cotton WRKY1 mediates the plant defense-to-development transition

767

during infection of cotton by Verticillium dahliae by activating JASMONATE

768

ZIM-DOMAIN1 expression. Plant Physiol 166, 2179-2194.

769

Li, R., Weldegergis, B.T., Li, J., Jung, C., Qu, J., Sun, Y., Qian, H., Tee, C., van Loon,

770

J.J., Dicke, M., et al. (2014b). Virulence factors of geminivirus interact with MYC2 to

771

subvert plant resistance and promote vector performance. Plant Cell 26, 4991-5008.

772

Li, Y.B., Han, L.B., Wang, H.Y., Zhang, J., Sun, S.T., Feng, D.Q., Yang, C.L., Sun,

773

Y.D., Zhong, N.Q., and Xia, G.X. (2016). The Thioredoxin GbNRX1 plays a crucial

774

role in homeostasis of apoplastic reactive oxygen species in response to Verticillium

775

dahliae infection in cotton. Plant Physiol 170, 2392-2406. 37

776

Lian, T.F., Xu, Y.P., Li, L.F., and Su, X.D. (2017). Crystal structure of tetrameric

777

Arabidopsis MYC2 reveals the mechanism of enhanced interaction with DNA. Cell

778

Rep 19, 1334-1342.

779

Liang, X., Ding, P., Lian, K., Wang, J., Ma, M., Li, L., Li, L., Li, M., Zhang, X., Chen,

780

S., et al. (2016). Arabidopsis heterotrimeric G proteins regulate immunity by directly

781

coupling to the FLS2 receptor. Elife 5, e13568.

782

Liu, T.L., Song, T.Q., Zhang, X., Yuan, H.B., Su, L.M., Li, W.L., Xu, J., Liu, S.H.,

783

Chen, L.L., Chen, T.Z., et al. (2014). Unconventionally secreted effectors of two

784

filamentous pathogens target plant salicylate biosynthesis. Nat Commun 5, 4686.

785

Liu, Y., Schiff, M., and Dinesh-Kumar, S.P. (2002). Virus-induced gene silencing in

786

tomato. Plant J 31, 777-786.

787

Lo Presti, L., Lanver, D., Schweizer, G., Tanaka, S., Liang, L., Tollot, M., Zuccaro, A.,

788

Reissmann, S., and Kahmann, R. (2015). Fungal effectors and plant susceptibility.

789

Annu Rev Plant Biol 66, 513-545.

790

Nawrath, C., Heck, S., Parinthawong, N., and Metraux, J.P. (2002). EDS5, an

791

essential component of salicylic acid-dependent signaling for disease resistance in

792

Arabidopsis, is a member of the MATE transporter family. Plant Cell 14, 275-286.

793

Nomura, K., Mecey, C., Lee, Y.N., Imboden, L.A., Chang, J.H., and He, S.Y. (2011).

794

Effector-triggered immunity blocks pathogen degradation of an immunity-associated

795

vesicle traffic regulator in Arabidopsis. Proc Natl Acad Sci USA 108, 10774-10779.

796

Ottmann, C., Luberacki, B., Kufner, I., Koch, W., Brunner, F., Weyand, M., Mattinen,

797

L., Pirhonen, M., Anderluh, G., Seitz, H.U., et al. (2009). A common toxin fold 38

798

mediates microbial attack and plant defense. Proc Natl Acad Sci USA 106,

799

10359-10364.

800

Qin, J., Zhao, J.Y., Zuo, K.J., Cao, Y.F., Ling, H., Sun, X.F., and Tang, K.X. (2004).

801

Isolation and characterization of an ERF-like gene from Gossypium barbadense. Plant

802

Sci 167, 1383-1389.

803

Santhanam, P., and Thomma, B.P.H.J. (2013). Verticillium dahliae Sge1 differentially

804

regulates expression of candidate effector genes. Mol Plant Microbe Interact 26,

805

249-256.

806

Santhanam, P., van Esse, H.P., Albert, I., Faino, L., Nurnberger, T., and Thomma,

807

B.P.H.J. (2013). Evidence for functional diversification within a fungal NEP1-like

808

protein family. Mol Plant Microbe Interac 26, 278-286.

809

Schaible, L., Cannon, O.S., and Waddoups, V. (1951). Inheritance of resistance to

810

Verticillium wilt in a tomato cross. Phytopathology 41, 986-990.

811

Schnathorst, W.C. (1981). Life cycle and epidemiology of Verticillium. pp. 81-111.

812

Schornack, S., van Damme, M., Bozkurt, T.O., Cano, L.M., Smoker, M., Thines, M.,

813

Gaulin, E., Kamoun, S., and Huitema, E. (2010). Ancient class of translocated

814

oomycete effectors targets the host nucleus. Proc Natl Acad Sci USA 107,

815

17421-17426.

816

Spanu, P.D., Abbott, J.C., Amselem, J., Burgis, T.A., Soanes, D.M., Stuber, K., van

817

Themaat, E.V.L., Brown, J.K.M., Butcher, S.A., Gurr, S.J., et al. (2010). Genome

818

expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme

819

parasitism. Science 330, 1543-1546. 39

820

Sun, L.Q., Zhu, L.F., Xu, L., Yuan, D.J., Min, L., and Zhang, X.L. (2014). Cotton

821

cytochrome P450 CYP82D regulates systemic cell death by modulating the

822

octadecanoid pathway. Nat Commun 5, 5372.

823

Sun, T.J., Busta, L., Zhang, Q., Ding, P., Jetter, R., and Zhang, Y.L. (2018).

824

TGACG-BINDING FACTOR 1 (TGA1) and TGA4 regulate salicylic acid and

825

pipecolic acid biosynthesis by modulating the expression of SYSTEMIC ACQUIRED

826

RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g

827

(CBP60g). New Phytol 217, 344-354.

828

Sun, T.J., Zhang, Y.X., Li, Y., Zhang, Q., Ding, Y.L., and Zhang, Y.L. (2015).

829

ChIP-seq reveals broad roles of SARD1 and CBP60g in regulating plant immunity.

830

Nat Commun 6, 10159.

831

Tian, H., Zhou, L., Guo, W.Z., and Wang, X.Y. (2015). Small GTPase Rac1 and its

832

interaction partner Cla4 regulate polarized growth and pathogenicity in Verticillium

833

dahliae. Fungal Genet Biol 74, 21-31.

834

Tzima, A., Paplomatas, E.J., Rauyaree, P., and Kang, S. (2010). Roles of the catalytic

835

subunit of cAMP-dependent protein kinase A in virulence and development of the

836

soilborne plant pathogen Verticillium dahliae. Fungal Genet Biol 47, 406-415.

837

Tzima, A.K., Paplomatas, E.J., Rauyaree, P., Ospina-Giraldo, M.D., and Kang, S.

838

(2011). VdSNF1, the sucrose nonfermenting protein kinase gene of Verticillium

839

dahliae, is required for virulence and expression of genes involved in cell-wall

840

degradation. Mol Plant Microbe Interac 24, 129-142.

841

Tzima, A.K., Paplomatas, E.J., Tsitsigiannis, D.I., and Kang, S. (2012). The G protein 40

842

b subunit controls virulence and multiple growth- and development-related traits in

843

Verticillium dahliae. Fungal Genet Biol 49, 271-283.

844

Vallad, G.E., and Subbarao, K.V. (2008). Colonization of resistant and susceptible

845

lettuce cultivars by a green fluorescent protein-tagged isolate of Verticillium dahliae.

846

Phytopathology 98, 871-885.

847

Wang, L., Tsuda, K., Sato, M., Cohen, J.D., Katagiri, F., and Glazebrook, J. (2009).

848

Arabidopsis CaM binding protein CBP60g contributes to MAMP-induced SA

849

accumulation and is involved in disease resistance against Pseudomonas syringae.

850

PLoS Pathog 5, e1000301.

851

Wang, L., Tsuda, K., Truman, W., Sato, M., Nguyen, L.V., Katagiri, F., and

852

Glazebrook, J. (2011). CBP60g and SARD1 play partially redundant critical roles in

853

salicylic acid signaling. Plant J 67, 1029-1041.

854

Wang, S., Xing, H.Y., Hua, C.L., Guo, H.S., and Zhang, J. (2016). An improved

855

single-step cloning strategy simplifies the Agrobacterium tumefaciens-mediated

856

transformation (ATMT)-based gene-disruption method for Verticillium dahliae.

857

Phytopathology 106, 645-652.

858

Whisson, S.C., Boevink, P.C., Moleleki, L., Avrova, A.O., Morales, J.G., Gilroy, E.M.,

859

Armstrong, M.R., Grouffaud, S., van West, P., Chapman, S., et al. (2007). A

860

translocation signal for delivery of oomycete effector proteins into host plant cells.

861

Nature 450, 115-118.

862

Wildermuth, M.C., Dewdney, J., Wu, G., and Ausubel, F.M. (2001). Isochorismate

863

synthase is required to synthesize salicylic acid for plant defence. Nature 414, 41

864

562-565.

865

Xu, L., Zhang, W., He, X., Liu, M., Zhang, K., Shaban, M., Sun, L., Zhu, J., Luo, Y.,

866

Yuan, D., et al. (2014). Functional characterization of cotton genes responsive to

867

Verticillium dahliae through bioinformatics and reverse genetics strategies. J Exp Bot

868

65, 6679-6692.

869

Yang, C.L., Liang, S., Wang, H.Y., Han, L.B., Wang, F.X., Cheng, H.Q., Wu, X.M.,

870

Qu, Z.L., Wu, J.H., and Xia, G.X. (2015). Cotton major latex protein 28 functions as a

871

positive regulator of the ethylene responsive factor 6 in defense against Verticillium

872

dahliae. Mol Plant 8, 399-411.

873

Zhang, D.D., Wang, X.Y., Chen, J.Y., Kong, Z.Q., Gui, Y.J., Li, N.Y., Bao, Y.M., and

874

Dai, X.F. (2016). Identification and characterization of a pathogenicity-related gene

875

VdCYP1 from Verticillium dahliae. Sci Rep 6, 27979.

876

Zhang, J., Li, W., Xiang, T., Liu, Z., Laluk, K., Ding, X., Zou, Y., Gao, M., Zhang, X.,

877

Chen, S., et al. (2010a). Receptor-like cytoplasmic kinases integrate signaling from

878

multiple plant immune receptors and are targeted by a Pseudomonas syringae effector.

879

Cell Host Microbe 7, 290-301.

880

Zhang, L.S., Ni, H., Du, X., Wang, S., Ma, X.W., Nurnberger, T., Guo, H.S., and Hua,

881

C.L. (2017). The Verticillium-specific protein VdSCP7 localizes to the plant nucleus

882

and modulates immunity to fungal infections. New Phytol 215, 368-381.

883

Zhang, Y.X., Xu, S.H., Ding, P.T., Wang, D.M., Cheng, Y.T., He, J., Gao, M.H., Xu, F.,

884

Li, Y., Zhu, Z.H., et al. (2010b). Control of salicylic acid synthesis and systemic

885

acquired resistance by two members of a plant-specific family of transcription factors. 42

886

Proc Natl Acad Sci USA 107, 18220-18225.

887

Zhao, P., Zhao, Y.L., Jin, Y., Zhang, T., and Guo, H.S. (2014). Colonization process of

888

Arabidopsis thaliana roots by a green fluorescent protein-tagged isolate of

889

Verticillium dahliae. Protein Cell 5, 94-98.

890

Zhao,

891

VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by

892

Verticillium dahliae. PLoS Pathog 12, e1005793.

893

Zhou, B.J., Jia, P.S., Gao, F., and Guo, H.S. (2012). Molecular characterization and

894

functional analysis of a necrosis- and ethylene-inducing, protein-encoding gene

895

family from Verticillium dahliae. Mol Plant Microbe Interac 25, 964-975.

896

Zhou, T.T., Zhao, Y.L., and Guo, H.S. (2017). Secretory proteins are delivered to the

897

septin-organized penetration interface during root infection by Verticillium dahliae.

898

PloS Pathog 13, e1006275.

Y.L.,

Zhou,

T.T.,

and

Guo,

H.S.

(2016).

Hyphopodium-specific

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Figure Legends

901

Figure 1. VdSCP41 contributes to V. dahliae virulence on host plants.

902

(A) Schematic description of the generation of the VdΔscp41 mutant. (B) Southern

903

blot analysis of the VdSCP41 gene deletion in the VdΔscp41 mutant. Genomic DNA

904

samples isolated from V592 and VdΔscp41 strains were digested by BamHI and

905

subjected to Southern blot analysis. (C-D) Disease symptoms of upland cotton (C)

906

and Arabidopsis (D) plants infected with the wildtype (V592), VdΔscp41, and

907

VdΔscp41/VdSCP41-GFP strains, as indicated. (E-F) Disease index analyses of 43

908

upland cotton (E) and Arabidopsis (F) infected with the indicated strains. The plants

909

were photographed and subjected to disease index analyses 3-4 weeks post

910

inoculationThe disease indexes were evaluated with three replicates generated from

911

24 plants for each inoculum. Error bars indicate standard deviation of three biological

912

replicates. Student’s t test was carried out to determine the significance of difference.

913

* indicates significant difference at P value of